Optimization of a flow cytometric assay to evaluate the human neutrophil ability to phagocytose immune complexes via Fc gamma and complement receptors

Autor(es)

Santos, Everton O. L. ^{[1, 2]} ; Azzolini, Ana Elisa C. S. ^[2] ; Lucisano-Valim, Yara M. ^[2]

Afiliação do(s) autor(es)

^[1] Univ Sao Paulo, Fac Ciencias Farmaceut Ribeirao Preto, Dept Bioquim & Imunol, BR-14040903 Ribeirao Preto, SP - Brazil

^[2] Univ Sao Paulo, Fac Ciencias Farmaceut Ribeirao Preto, Dept Quim & Fis, BR-14040903 Ribeirao Preto, SP - Brazil

• Tipo de documento: Artigo Científico
• Fonte: JOURNAL OF PHARMACOLOGICAL AND TOXICOLOGICAL METHODS; v. 72, p. 67-71, MAR-APR 2015.
• Citações Web of Science: 6

Resumo

Introduction: This study aims to optimize some experimental conditions of a flow cytometric assay to examine the human neutrophil ability to phagocytose immune complexes (ICs) via Fc gamma and complement receptors (Fc gamma R and CR, respectively). The parameters assessed were: number of cells, concentration of ICs, reaction time, pH and concentration of the Trypan Blue quenching solution. Methods: Neutrophils were isolated from peripheral blood of healthy volunteers. Precipitated ICs composed of IgG and fluorescein isothiocyanate (FITC)-labeled ovalbumin, opsonized or not with serum complement, were used to trigger the neutrophil phagocytosis via Fc gamma R, CR, and Fc gamma R + CR. Fluorescence of the internalized ICs was measured by flow cytometry, after quenching the extracellular fluorescence with Trypan Blue. Results: The optimal experimental conditions established for the phagocytosis assay were: 1 x 10(6) cells mL(-1) and 40 mu g mL(-1) FITC-labeled ICs, incubated for 30 min, at 37 degrees C, in 0.5 mL of reaction volume. Trypan Blue solution at 1.25 mg mL(-1) pH 4.4 was the best fluorescence quencher of FITC-labeled ICs attached to the outer surface of neutrophils. Discussion: The selected experimental conditions were viable to evaluate IC phagocytosis by neutrophils; they are also suitable to compare the efficiency of IC phagocytosis mediated by Fc gamma R and CR classes of membrane receptors, alone or in combination. This method finds application in studies of (i) the receptor-specific phagocytic function of normal and pathogenic neutrophils as well as (ii) the impact of drugs and therapies on this essential effector function of neutrophils. (C) 2014 Elsevier Inc. All rights reserved. (AU)

• Processo FAPESP: 07/06925-5 - Mecanismo de ação de flavonóides no metabolismo oxidativo e na fagocitose de neutrófilos humanos desencadeados por receptores FC gamma e Cr
• Beneficiário: Everton de Oliveira Lima dos Santos
• Modalidade de apoio: Bolsas no Brasil - Mestrado
• Processo FAPESP: 07/02487-3 - Estudo do mecanismo de ação de derivados cumarínicos sobre a atividade da mieloperoxidase: investigação da metabolização dessas substâncias a radicais livres fenoxílicos, implicações no estresse oxidativo e na modulação de funções de leucócitos
• Beneficiário: Yara Maria Lucisano Valim
• Modalidade de apoio: Auxílio à Pesquisa - Regular